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Enrichment of calcifying extracellular vesicles using density-based ultracentrifugation protocol
CitationHutcheson, Joshua D., Claudia Goettsch, Tan Pham, Masaya Iwashita, Masanori Aikawa, Sasha A. Singh, and Elena Aikawa. 2014. “Enrichment of calcifying extracellular vesicles using density-based ultracentrifugation protocol.” Journal of Extracellular Vesicles 3 (1): 10.3402/jev.v3.25129. doi:10.3402/jev.v3.25129. http://dx.doi.org/10.3402/jev.v3.25129.
Published Versiondoi:10.3402/jev.v3.25129
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ORIGINAL RESEARCH ARTICLE
Enrichment of calcifying extracellular vesicles usingdensity-based ultracentrifugation protocol
Joshua D. Hutcheson$, Claudia Goettsch$, Tan Pham, Masaya Iwashita,Masanori Aikawa, Sasha A. Singh and Elena Aikawa*
Center for Interdisciplinary Cardiovascular Sciences, Division of Cardiovascular Medicine, Brigham andWomen’s Hospital, Harvard Medical School, Boston, MA, USA
Calcifying extracellular vesicles (EVs) released from cells within atherosclerotic plaques have received
increased attention for their role in mediating vascular calcification, a major predictor of cardiovascular
morbidity and mortality. However, little is known about the difference between this pathologic vesicle
population and other EVs that contribute to physiological cellular processes. One major challenge that
hinders research into these differences is the inability to selectively isolate calcifying EVs from other vesicle
populations. In this study, we hypothesized that the formation of mineral within calcifying EVs would
increase the density of the vesicles such that they would pellet at a faster rate during ultracentrifugation.
We show that after 10 min of ultracentrifugation at 100,000�g, calcifying EVs are depleted from the
conditioned media of calcifying coronary artery smooth muscle cells and are enriched in the pelleted
portion. We utilized mass spectrometry to establish functional proteomic differences between the calcify-
ing EVs enriched in the 10 min ultracentrifugation compared to other vesicle populations pre-
ferentially pelleted by longer ultracentrifugation times. The procedures established in this study will allow
us to enrich the vesicle population of interest and perform advanced proteomic analyses to find subtle
differences between calcifying EVs and other vesicle populations that may be translated into therapeutic
targets for vascular calcification. Finally, we will show that the differences in ultracentrifugation times
required to pellet the vesicle populations can also be used to estimate physical differences between the
vesicles.
Keywords: calcification; extracellular vesicles; atherosclerosis; ultracentrifugation; isolation
Responsible Editor: Paul Harrison, University of Birmingham, United Kingdom.
*Correspondence to: Elena Aikawa, 3 Blackfan St., CLSB, CICS, 17th floor, Brigham and Women’s Hospital,
Harvard Medical School, Boston, MA 02115, USA, Email: eaikawa@rics.bwh.harvard.edu
Received: 6 June 2014; Revised: 7 November 2014; Accepted: 10 November 2014; Published: 5 December 2014
Amajor challenge in the study of extracellular
vesicles (EVs) is the difficulty in isolating popula-
tions of interest within the myriad of vesicle types
present in biological samples (1�3). Exosomes, micropar-
ticles, apoptotic bodies and matrix vesicles are all believed
to arise through different cellular mechanisms to perform
varying biological functions (1,4,5); however, significant
overlap exists in the size and structure of these vesicle
subtypes (6,7). Ultracentrifugation is a common technique
to isolate all vesicles from samples (3). This isolation is
usually followed by common assay techniques to identify
changes in RNA or protein expression. However, this
non-discriminatory technique is not suitable to identify
changes that occur in a vesicle subtype that represents
a small portion of the total vesicle population (3). To
overcome this problem, specific vesicles of interest are
often isolated using an immunoprecipitation-based tech-
nique, whereby antibodies against a known antigen on
specific vesicles are adsorbed to a larger bead that can be
isolated from the solution via magnetic or centrifuga-
tion methods (8,9). While this technique can be utilized
effectively, it requires a priori knowledge of differential
protein expression on the population of interest and there-
fore is not suitable for finding novel vesicle subpopula-
tions. Further, the expense associated with antibodies and
reagents required for immunoprecipitation approaches
$These authors contributed equally to the work.
�
Journal of Extracellular Vesicles 2014. # 2014 Joshua D. Hutcheson et al. This is an Open Access article distributed under the terms of the CreativeCommons Attribution-Noncommercial 3.0 Unported License (http://creativecommons.org/licenses/by-nc/3.0/), permitting all non-commercial use,distribution, and reproduction in any medium, provided the original work is properly cited.
1
Citation: Journal of Extracellular Vesicles 2014, 3: 25129 - http://dx.doi.org/10.3402/jev.v3.25129(page number not for citation purpose)
can be prohibitive for large-scale experiments or those in
which many replicates are required.
One specific vesicle subtype of interest to our research is
the calcifying EV, also termed as matrix vesicles (10,11).
Matrix vesicles have been well-described in bone develop-
ment, wherein osteoblast-derived vesicles nucleate hydroxy-
apatite crystals along collagen fibres in the developing
bone (12,13). Recently, calcifying EVs derived from mac-
rophages and smooth muscle cells (SMCs) have received
increased attention for their role in vascular calcification
(10,11,14�17). Matrix vesicles serve as nucleating foci
for the formation of microcalcifications within athero-
sclerotic plaque fibrous caps, which leads to plaque insta-
bility, rupture and subsequent myocardial infarction
and stroke (18�21). Given the observation that calcifying
matrix vesicles have been shown to exhibit an electron
dense structure as hydroxyapatite nucleation proceeds
(22), we hypothesized that time-dependent ultracentrifu-
gation may be used to enrich calcifying EVs with a greater
physical density than other vesicular populations from
vascular SMCs. Isolating EVs of interest improves the
sensitivity of assays by reducing background from other
vesicle subtypes, and herein, we will show that selective
enrichment of calcifying EVs enhances the signal-to-noise
ratio in various calcification and proteomic assays by
reducing the contribution from non-calcifying vesicles.
Utilizing this protocol will allow us to better characterize
the biophysical and proteomic properties of calcifying
EVs and may help identify potential therapeutic targets
for vascular calcification.
Methods
Cell culture and conditioned media collectionHuman coronary artery (SMCs, PromoCell) were grown
to confluence and were cultured in control media consist-
ing of DMEM with 10% (v/v) foetal bovine serum and
1% (v/v) penicillin/streptomycin (control) or a calcifying
media consisting of control media supplemented with
10 nM dexamethasone, 100 mM L-ascorbic acid and 10 mM
b-glycerophosphate. The media were replaced every 2 days.
The SMCs were cultured for at least 14 days, a time point
sufficient to induce osteogenic differentiation of the cells
in the calcifying media (23). For specified samples, an
inhibitor for tissue non-specific alkaline phosphatase
(TNAP) was used at a concentration of 1 mM (Calbio-
chem, #613810). After the prescribed culture period, the
culture media were replaced with media containing the
same components but lower foetal bovine serum (0.1%).
This was done to reduce the noise caused by the presence
of vesicles within the serum compared to the vesicles of
interest released by the SMCs. After 24 h, the low serum
media were collected after centrifugation at 1,000�g for
5 min to remove potential cellular contaminants. The
resulting supernatant was then stored at �808C prior
to further processing.
RNA preparation and real-time PCRTotal RNA from the cell culture was isolated using TriZol
(Life Technologies). Reverse transcription was performed
using the QuantiTect Reverse Transcription Kit (Qiagen,
Hilden, Germany). The mRNA expression was deter-
mined by TaqMan-based real-time PCR reactions (Life
Technologies). The following TaqMan probes were used:
Hs01029144_m1 (human ALPL) and 4326315E (human
b-actin). The expression levels were normalized to b-actin.
Results were calculated using the DDCt method, and
presented as fold increase relative to control.
Alizarin red S staining for calcium depositionCalcium deposition was assessed by Alizarin red S stain-
ing. SMCs were fixed in 10% formalin and stained with
40 mM Alizarin Red S (pH 4.2) for 30 min at room tem-
perature. Excess dye was removed by washing the plates
with distilled water. The extent of calcium mineral deposi-
tion is proportional to the amount of Alizarin red S stain
remaining in the culture well after washing.
Ultracentrifugation isolation of EVsSamples were thawed on ice, and 50 ml of each sample
was collected for nanoparticle tracking analysis (NTA)
prior to ultracentrifugation. Of the remaining sample,
1 ml was pipetted into 11�34 mm ultracentrifuge tubes
(Beckman Coulter). The tubes were loaded into a fixed
angle rotor (Beckman Coulter TLA 120.2) for ultracen-
trifugation (Beckman Coulter Optima MAX-UP Ultra-
centrifuge) at 100,000�g at 48C for 10 min. Following
this 10 min ultracentrifugation, 50 ml of the supernatant
was collected for NTA. The remaining supernatant was
transferred to a new ultracentrifugation tube and the
samples were subjected to an additional 20 min of ultracen-
trifugation at 100,000�g. The collection process, super-
natant transfer and ultracentrifugation protocol was
repeated for a 30 min ultracentrifugation followed by
a final sample collection and ultracentrifugation for 40 min.
The pellets from each ultracentrifugation collection time
point were washed once by re-suspending the pellets
in PBS and using ultracentrifugation at 100,000�g for
40 min to obtain the final pellet for further analyses.
NanoSight NTANanoSight NTA (NanoSight LM10, Malvern Instru-
ments Ltd, Malvern, UK) was used to assess vesicle size
and concentration within the media samples (24). The
samples were diluted 1:10 in PBS prior to injection into
the NanoSight chamber. For each sample, 2 NTA videos
were collected for 1 min apiece, and fresh media were
injected between the 2 recordings. The camera gain was
set at a constant value of 13, and the threshold value
for vesicle detection was set at 4. The resultant size and
Joshua D. Hutcheson et al.
2(page number not for citation purpose)
Citation: Journal of Extracellular Vesicles 2014, 3: 25129 - http://dx.doi.org/10.3402/jev.v3.25129
concentration output data were averaged to generate the
final distribution for each sample.
Alkaline phosphatase activityTo measure TNAP activity, cells or ultracentrifugation
pellets were re-suspended in 120 ml of TNAP Assay Buffer
(BioVision) and incubated overnight at �308C. TNAP
activity was measured using the BioVision Alkaline
Phosphatase Activity Colorimetric Assay kit according
to the manufacturer’s protocol (BioVision).
EV mineral contentTo measure calcium phosphate mineral content within
isolated EVs, we modified the fluorescence-based Osteo-
Image assay (Lonza) used for detection of hydroxyapatite
mineral in cell culture. The ultracentrifugation pellets
from 1 ml conditioned media were re-suspended and
fixed in 10% formalin for 15 min. The vesicles were then
washed once in PBS using 100,000�g ultracentrifuga-
tion for 40 min. Following this wash, the pellets were re-
suspended in 200 ml diluted OsteoImage dye (according
to manufacturer protocol) for 30 min at room tempera-
ture. The dye was then removed via ultracentrifugation
and the vesicles were washed twice in OsteoImage wash
buffer using 100,000�g ultracentrifugation for 40 min.
After the final wash, the pellet was re-suspended in 100 ml
OsteoImage wash buffer and fluorescence intensity was
measured using a plate reader. The resultant fluorescence
values were normalized to the number of vesicles pelleted
by ultracentrifugation (obtained from NanoSight) to ac-
count for variations in vesicle numbers in different treat-
ment conditions. Fold changes of these normalized values
for calcific vesicles compared to control conditions are
reported for each ultracentrifugation time point.
Transmission electron microscopyUltracentrifuged vesicle pellets were immersion fixed
in 2.5% glutaraldehyde, 2% paraformaldehyde, in 0.1 M
cacodylate buffer pH 7.4 [modified Karnovsky’s fixative
(25)] for at least 1 h at room temperature before being spun
down again and embedded in 4% low melting agarose.
Pellets were cut to size and placed at 48C overnight. To
continue processing, pellets were washed 3�5 min each in
PBS then fixed secondarily in 1% osmium tetroxide in
buffer, for 1 h at 48C. Pellets were then washed 3�5 min
in PBS, then 3�5 min in deionized water before being
taken through a dehydration series including 30, 50, 70,
95 and 100% ethanol, 10 min each at 48C. Pellets were then
brought to room temperature to continue dehydrating
with 100% ethanol (3�10 min each). Infiltration pro-
ceeded with 2:1 100% ethanol to LX112 Epon resin for
30 min followed by a 1:1 mixture of these components
overnight with the pellets rotated at room temperature.
The following day, the sample containers’ caps were left
open to evaporate the ethanol, and the remaining resin
was removed prior to the addition of 100% freshly
made resin. Samples were recapped and rotated overnight.
Before embedding the next day, samples were placed in a
vacuum oven at 608C for 2 h. Samples were embedded in
flat-bottomed BEEM capsules and cured at 608C for 48 h.
Cured blocks were sectioned using a Leica Ultracut E
ultramicrotome with a diamond knife at 80 nm thickness
and placed on bare 200 mesh copper grids. Grids were
contrast stained with 2% uranyl acetate for 10 min and
lead citrate for 5 min. Grids were imaged on a JEOL 1400
TEM equipped with a side mount Gatan Orius SC1000
digital camera.
Mass spectrometryFor mass spectrometry analysis of the pelleted vesicles,
the vesicles were lysed in RIPA buffer, and polyacrylamide
gel electrophoresis was used to separate the proteins by
size. The polyacrylamide gels were stained with coomassie
blue to visualize the separated protein bands. The gels
were then cut into small strips dictated by the appearance
of protein bands that exhibited differences between the
treatment groups. Trypsin was used to digest the proteins
into peptide sequences prior to mass spectrometry. Peptide
samples were analyzed with the high resolution/accuracy
LTQ-Orbitrap (Elite model) mass spectrometer fronted
with a Nanospray FLEX ion source, and coupled to
an Easy-nLC1000 HPLC pump (Thermo Scientific). The
peptides were subjected to a dual column set-up: an
Acclaim PepMap RSLC C18 trap column, 75 mm�20 mm;
and an analytical column packed in-house with 3 mm,
100 A pore size C18 resin (Bruker) in a 75 mm�200 mm
long piece of fused silica capillary. The analytical gra-
dient was run at 350 nl/min from 10 to 30% Solvent B
(acetonitrile/0.1% formic acid) for 30 min followed by
5 min of 95% Solvent B. Solvent A was 0.1% formic acid.
All reagents were HPLC-grade. The instrument was set
at 120 K resolution, and the top 20 precursor ions (within a
scan range of 380�2,000 m/z) were subjected to collision-
induced dissociation (collision energy 35%) for peptide
sequencing (MS/MS). The MS/MS data were queried
against the Human UniProt database (downloaded on
27 March 2012) using the SEQUEST search algorithm
(26) via the Proteome Discoverer Package (version 1.4,
Thermo Scientific) using methionine oxidation as a vari-
able modification, and carbamidomethylation of cysteine
residues as a fixed modification. The peptide false dis-
covery rate (FDR) was calculated using Percolator pro-
vided by Proteome Discoverer: the FDR was determined
based on the number of MS/MS spectral hits when searched
against the reverse, decoy Human database (27,28). Peptides
were filtered based on a 1% FDR. Proteins used for
spectral quantification contained ]2 unique peptides.
Statistical analysisAll quantitative data are presented as mean values from
at least 6 independent experiments (n�6) with error bars
representing the standard deviation of the mean. Unless
Enrichment of calcifying extracellular vesicles
Citation: Journal of Extracellular Vesicles 2014, 3: 25129 - http://dx.doi.org/10.3402/jev.v3.25129 3(page number not for citation purpose)
otherwise noted, differences between control and calcify-
ing treatment groups were determined using unpaired
t-tests with the statistical significance threshold set as 0.05
(i.e. pB0.05). The mass spectrometry data were generated
using the combined data from 2 independent experiments.
Results
Osteogenic reprogramming and calcium mineraldepositionSMCs in calcifying media exhibit a significant 2.8-fold
increase in TNAP activity after 7 days in culture that
increases to 5-fold after 21 days in culture (Fig. 1a). This
elevated TNAP activity is concomitant with an increase
in mineralization as shown by Alizarin red S staining
(Fig. 1b). The increased TNAP activity was found to cor-
respond to a significant increase in TNAP mRNA ex-
pression after 14 days in the calcifying SMC (Fig. 1c).
Treating SMCs with 1 mM TNAP inhibitor abrogated the
mineralization observed after 21 days in culture (Fig. 1d).
Ultracentrifugation enrichment of calcifying matrixvesiclesNTAs revealed similar size profiles for EVs released from
SMCs cultured in control or calcifying media for greater
than 14 days (Fig. 2a). The majority of the measured
vesicles for both groups were between 30 and 300 nm
in diameter, consistent with previous literature values for
exosomes, matrix vesicles and microparticles (1). Vesicles
released from calcifying SMCs exhibited a trending but
insignificant increase in mean vesicle size (185917) com-
pared to vesicles from control cultures (175916 nm,
p�0.2); however, the concentration of vesicles measured
in the conditioned media from calcifying SMCs was sig-
nificantly higher compared to control SMCs (Fig. 2b).
After 10 min ultracentrifugation at 100,000�g, the
vesicle concentration between the 2 treatment groups was
no longer different (Fig. 2c). A pair-wise comparison
revealed a significant difference in the initial rate (i.e. the
slope calculated from the initial vesicle concentration to
the vesicle concentration remaining after 10 min) of pel-
leted vesicles (Fig. 2d) during the first 10 min (calcifying:
39921 vesicles per minute, control: 28917 vesicles per
minute, p�0.049). No quantifiable difference in concen-
tration was found for all subsequent ultracentrifugation
times studied. Pair-wise tests were used for this analysis
due to the variability in initial vesicle concentrations from
the SMC donors and the desire to measure the rate of
vesicles pelleted from the same donor samples over time.
Control
OM
Day 7 Day 14 Day 21
a) b)Cellular TNAP
Control
8
6
4
2
00
6
4
2
0
7 14 21
OM
Day
§
**
*
Fol
d in
crea
seF
old
incr
ease
c) ALPL mRNA
Control OM
*
d)Control OM
DMSO
TNAPInhibitor
Fig. 1. Osteogenic transition of vascular SMCs. a) Time-dependent TNAP activity. §�pB0.05 by ANOVA. b) Representative images
of Alizarin Red S stained mineralized matrix at day 21. c) ALP mRNA at day 14. d) Representative images of Alizarin Red S stained
mineralized matrix at day 21 after treatment with TNAP inhibitor (1 mM). *�pB0.05 control versus OM. n�4 independent
experiments. Mean9SD.
Joshua D. Hutcheson et al.
4(page number not for citation purpose)
Citation: Journal of Extracellular Vesicles 2014, 3: 25129 - http://dx.doi.org/10.3402/jev.v3.25129
Time, min
Time, min
1000
800
600
400
200
60
40
20
0
00 20 40 60 80 100
0 20 40 60 80 100Time, min
0 20 40 60 80 100
x10
6/m
l
Control
OM
Control
OM
Control
OM
c) Vesicle concentration0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.02
Vesicle protein concentration
a) 5
4
3
2
1
0
5
6
4
3
2
1
0
0 200 400 600
Nanoparticle tracking analyses
Vesicles diameter, nm
Nu
mb
er,
x1
06/m
l
Control
x10
6/m
l
Vesicle concentrationb)
Control
OM
d)
f)e)
Ve
sicl
es/
min
Initial pellet rate
Vesicle protein pelleting rate
1000
800
600
400
200
0
OM
Control OM
*
*
**
*µ
g/m
l/min
µg
/ml
Fig. 2. Ultracentrifugation of extracellular vesicles. a) NanoSight NTA plot of vesicle diameter versus concentration for control and
calcifying samples. b) The concentration of vesicles in the conditioned media of control and calcifying samples as measured by NTA.
Connecting lines indicate data obtained from the same experiment. c) The concentration of vesicles remaining in the conditioned media
samples over the ultracentrifugation time course. d) The rate at which vesicles are pelleted during the first 10 min of ultracentrifugation.
Connecting lines indicate data obtained from the same experiment. e) Protein assay of pelleted vesicles over the ultracentrifugation time
course. f) The rate of pelleted protein over the ultracentrifugation time course. *�pB0.05. n�8 independent experiments. Mean9SD.
Enrichment of calcifying extracellular vesicles
Citation: Journal of Extracellular Vesicles 2014, 3: 25129 - http://dx.doi.org/10.3402/jev.v3.25129 5(page number not for citation purpose)
Similar to the NTA measurements, BCA protein analyses
revealed significantly higher amounts of protein in the
10 min ultracentrifugation pellet of vesicles from calcify-
ing SMCs compared to the protein pelleted from con-
trol vesicles (Fig. 2e). Each subsequent centrifugation
time after 10 min showed no difference in pelleted protein
between control and calcifying samples. Dividing the pro-
tein measured in the pellet at each time point by the
ultracentrifugation time elapsed since the previous time
point yields the rate of protein accumulation in the pellet
for each time point considered (Fig. 2f). The rate of
pelleted protein per minute was significantly higher in
the calcifying samples compared to control samples only
over the first 10 min of ultracentrifugation.
Calcification potential of isolated vesicle populationsTNAP is a common marker of osteogenic potential and
its presence in osteoblast-derived matrix vesicles has been
shown to confer calcification potential to the vesicles (22).
Therefore, TNAP activity was assayed in the pelleted
fraction of each ultracentrifugation time point as a
measure of vesicle calcification potential (Fig. 3a). The
10 min ultracentrifugation pellet from osteogenic samples
exhibited significantly higher TNAP activity than the
pellet from control samples. The pellets in each subse-
quent time point showed no difference in TNAP activity
in osteogenic and control samples. Pelleted vesicles from
10 min ultracentrifugation of calcifying SMC media
exhibited a 4.6-fold increase in TNAP activity compared
to the control pellet (Fig. 3b). As a comparison of the
10 min ultracentrifugation with commonly used vesicle
isolation protocols that use longer ultracentrifugation
times, samples were subjected to 100,000�g ultracentri-
fugation for 40 min (without collecting pellets from earlier
time points). Using this protocol, the pelleted portion
from osteogenic samples displayed a 3.1-fold increase in
TNAP activity compared to the time-matched control
samples (Fig. 3b).
To measure the mineral content of the EVs, we utilized
OsteoImage-hydroxyapatite-specific fluorescent dye. EVs
from SMCs cultured in calcifying media pelleted by
10 min ultracentrifugation at 100,000�g exhibited an
8.9-fold higher fluorescent signal per vesicle than EVs
from SMCs cultured in control media (Fig. 3c). The EVs
obtained from ultracentrifugation of the 10 min super-
natant for an additional 30 min at 100,000�g exhibited
significantly less fluorescent signal per vesicle (p�0.03).
Only background fluorescence was observed when this
assay was performed on media not used in SMC culture.
To further assess changes in the isolated vesicle fractions,
we utilized TEM to image the contents of the ultracen-
trifugation pellets. TEM of 10 min pellets revealed vesi-
cular structures in control samples (Fig. 3d). The 10 min
ultracentrifugation pellet from calcifying conditioned
media exhibit electron dense structures associated with
hydroxyapatite crystals (Fig. 3e) that are reminiscent of
TEM images previously reported for calcifying matrix
vesicles from osteoblasts (29). The 30 min ultracentrifu-
gation pellet for both control (Fig. 3f) and calcifying
(Fig. 3g) samples contained vesicular structures closer in
appearance to the 10 min pellet from the control samples.
The hydroxyapatite-associated structures were not ob-
served in the 30 min ultracentrifugation pellets.
Mass spectrometry profile of calcifying matrixvesiclesMass spectrometry was used to perform an unbiased pro-
teomic analysis of control and calcifying media pelleted
portions from ultracentrifugation for 10 min, and from
the pellets resulting from an additional 20 min ultracen-
trifugation of the 10 min supernatant (30 total min).
A total of 422 proteins (with ]2 unique peptides) were
identified between the 2 conditions. The logarithm was
taken of the ratio of the spectral counts identified in
the calcifying samples versus control samples. We then
plotted these normalized spectral count values from the
proteins most enriched in the calcifying samples to those
most enriched in the control samples to visualize the
distribution of protein enrichment between these 2 treat-
ment groups. After 10 min ultracentrifugation, 48 proteins
were found exclusively in the pellets from the calci-
fying matrix vesicles, whereas only 27 proteins were found
exclusively in pellets from the control samples (Fig. 4a).
Further, a larger number of proteins were found to be
enriched in the 10 min ultracentrifugation pellet of the
calcifying samples versus the 10 min ultracentrifugation
pellet of the control samples. An example MS2 spectrum
of a known calcification marker, TNAP, is shown in
Fig. 4b. The logarithmic plot is marked to show proteins
with greater than 2-fold enrichment for each treatment
group. We used a gene ontology (GO) database to estab-
lish the functional annotation of the identified pro-
teins (Tables I and II). Proteins enriched in EVs isolated
from control media were found to be associated with
binding of extracellular matrix and matrix-associated
components (Table I), whereas proteins enriched in calci-
fying EVs were found to be associated with calcium ion
binding and enzymatic functions in addition to matrix
and protein binding (Table II). Specifically, our mass
spectrometric analyses identified 4 proteins previously
shown to be involved in EV-mediated vascular calcifica-
tion (Table III). In addition, among the proteins detected
in the 10 min pellet by mass spectrometry were a variety
of transmembrane proteins such as the common plasma
membrane protein, sodium/potassium ATPase, indicating
that this ultracentrifugation time was sufficient to pellet
membrane-derived vesicles.
Joshua D. Hutcheson et al.
6(page number not for citation purpose)
Citation: Journal of Extracellular Vesicles 2014, 3: 25129 - http://dx.doi.org/10.3402/jev.v3.25129
Discussion
Ultracentrifugation enrichment of calcifying matrixvesiclesOur results show an increase in the mineralization poten-
tial of vascular SMCs cultured in calcifying media. Min-
eral deposition was inhibited by the addition of an
inhibitor to TNAP, thus providing the impetus for us to
study the subset of calcifying EVs containing this enzyme.
In this study we show that the subpopulation of TNAP-
positive calcifying EV can be enriched from the total EV
population by taking advantage of the increased vesicle
density that results from the growth of mineral within the
vesicles. Our results indicate that after 10 min of ultracen-
trifugation at 100,000�g, the dense calcifying vesicles are
preferentially pelleted compared to the less dense control
vesicle population (Fig. 4c) as assessed by TNAP activity
and mineral content. Similar techniques could be used in
the future to enrich certain vesicle populations expected to
exhibit a physical difference from other vesicle populations.
The purpose of the current study was to take
advantage of the physical density change associated
Time, min
U/m
in/m
g pr
otei
n
Control
OM
Vesicle TNAP
*
b)a)
Time-matchedcontrol
Fol
d in
crea
se
Vesicle TNAP
10 min 40 min
*
*
d) e)
f) g)
OM
Nor
mal
ized
fluo
resc
ence
(OM
/Con
trol
)
10 min
*
c) Vesicle mineral content
30 min
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
40
30
20
10
0
0 20 40 60 80 100
8
6
4
2
0
Fig. 3. Calcification potential of pelleted vesicles. a) TNAP activity measured in the pelleted vesicles over the ultracentrifugation time
course. b) TNAP activity measured in conditioned media samples after 10 min ultracentrifugation or 40 min ultracentrifugation.
Control: time-matched control. Line indicates mean from 6 to 10 independent experiments. c) Normalized fluorescence intensity
(per vesicle) and fold increase OM/Control from 7 independent experiments. Line indicates mean. *�pB0.05 by Wilcoxon matched-
pairs signed-rank test. d) TEM images of vesicles from control or e) calcifying media pelleted at 10 min ultracentrifugation followed
by an additional 20 min ultracentrifugation for the same f) control and g) calcifying samples (30 min total ultracentrifugation).
*�pB0.05. n�6�7 independent experiments for A�C. Mean9SD.
Enrichment of calcifying extracellular vesicles
Citation: Journal of Extracellular Vesicles 2014, 3: 25129 - http://dx.doi.org/10.3402/jev.v3.25129 7(page number not for citation purpose)
Nor
mal
ized
LOG
10 (O
M/C
ontro
l)
Protein No.
Unique for OM
Unique for control
10 min
Cal
cify
ing
Con
trol
20 min
a)
c)
Greater than 2-foldenrichment in OM
0.3
–0.3Greater than 2-foldenrichment in control
No difference betweencontrol and OM
10 min 20 min
40 min
Step-wise centrifugation
40 minute centrifugation
b)
Transfer ofsupernatant
Transfer ofsupernatant
Inte
nsity
m/z
200 400 600 800 1000
TNAP
[M+2H]2+–NH3–H2O620.37
Protein No.
Unique for OM
Un
L D D D T W KG L L V
50 100 150 200
2
1
0
–1
–2
4000
3000
2000
1000
0
Fig. 4. Mass spectrometric profile of pelleted vesicles. a) Log plot of protein peptide spectral counts in calcifying vesicles versus control
vesicles after 10 min ultracentrifugation. b) An example MS2 spectrum of a known calcification marker, TNAP, is shown in Fig. 4b. c)
Schematic of serial ultracentrifugation protocol to enrich calcifying vesicles versus traditional ultracentrifugation protocol for 40 min.
Joshua D. Hutcheson et al.
8(page number not for citation purpose)
Citation: Journal of Extracellular Vesicles 2014, 3: 25129 - http://dx.doi.org/10.3402/jev.v3.25129
with mineralization of calcifying EVs from the larger
population of EVs. The method presented in this study
allowed us to detect TNAP activity and mineralization
in a subset of dense vesicles that were pelleted after only
10 min of ultracentrifugation. Our main goal was to
enrich this calcifying subset and reduce the noise caused
by other vesicle populations. Therefore, the current study
provides a template for calcifying vesicle enrichment but
does not address methods of obtaining a larger total
number of calcifying vesicles. A viable method to accom-
plish this goal would be to enhance the calcifying vesicle
signal by using collagenase digestion to isolate more cal-
cifying vesicles that become trapped in the extracellular
matrix and membrane microvilli during in vitro calcifi-
cation (30�32). Future studies may combine these 2
methods to both increase the signal due to more matrix
vesicles and decrease the noise caused by non-calcifying
vesicle populations.
Researchers should be aware that although ultracen-
trifugation is a non-specific means to isolate vesicles,
different ultracentrifugation parameters could produce
different results. As we have shown in this study, the cal-
cifying vesicles are completely removed from the condi-
tioned media after 10 min of ultracentrifugation (Fig. 3a).
By continuing the ultracentrifugation for longer times,
increasing numbers of other vesicle populations are also
pelleted. The presence of other vesicle populations in the
calcification analyses reduces the signal-to-noise ratio of
protein or gene-based assays because the other popula-
tions contribute to the total amount of protein or genetic
content assayed but do not increase the amount of the
calcification measurement. For example, in our study,
ultracentrifugation of the conditioned media for 40 min
instead of 10 min led to a decrease in the measured amount
of TNAP activity per milligram of protein (Fig. 3b). The
absolute amount of measured raw TNAP activity did not
change between these ultracentrifugation times; however,
the accumulation of vesicles with no TNAP activity in the
pellet resulted in the observed loss in signal due to an in-
crease in the amount of protein used to normalize the data.
Small but important changes could be missed by not
considering the effects of ultracentrifugation speed and
time on the vesicle populations that are isolated. In our
study, we used 2 independent experiments for our mass
spectrometry analyses. These 2 experiments were per-
formed using cultures of primary vascular SMCs obtained
from 2 independent donors. In Table III, the donor-to-
donor variability can be seen. Many proteins follow simi-
lar trends; however, the abundance and behaviour can
be slightly different between donors. This is always a
challenge when working with primary cells, but we believe
it is also a strength, as the results may be more reflective
of those obtained from the human population. In order
to better assess the variability between donors, technical
variation and noise must be minimized. Now that we have
a way to enrich our vesicles of interest, we can work to
understand the donor variability and the implications on
Table I. Functional annotation for proteins enriched in control
EV
GO term ID GO term
Number of
genes p
GO:0005539 Glycosaminoglycan
binding
3 3.17 E � 04
GO:0030247 Polysaccharide
binding
3 4.08 E � 04
GO:0001871 Pattern binding 3 4.08 E � 04
GO:0030246 Carbohydrate
binding
3 0.001919
GO:0050840 Extracellular matrix
binding
2 0.012363
Table II. Functional annotation for proteins enriched in calcifying EV
GO term ID GO term Number of genes p
GO:0005509 Calcium ion binding 21 4.32 E � 09
GO:0005198 Structural molecule activity 12 1.80 E � 04
GO:0004857 Enzyme inhibitor activity 8 2.88 E � 04
GO:0030246 Carbohydrate binding 7 0.006703
GO:0004866 Endopeptidase inhibitor activity 6 6.29 E � 04
GO:0030414 Peptidase inhibitor activity 6 8.02 E � 04
GO:0005178 Integrin binding 4 0.002787
GO:0004867 Serine-type endopeptidase inhibitor activity 4 0.009633
GO:0051082 Unfolded protein binding 4 0.017534
GO:0001871 Pattern binding 4 0.037223
GO:0030247 Polysaccharide binding 4 0.037223
GO:0001948 Glycoprotein binding 3 0.013423
GO:0030911 TPR domain binding 2 0.014259
GO:0030235 Nitric-oxide synthase regulator activity 2 0.023654
Enrichment of calcifying extracellular vesicles
Citation: Journal of Extracellular Vesicles 2014, 3: 25129 - http://dx.doi.org/10.3402/jev.v3.25129 9(page number not for citation purpose)
the production and calcification of EVs. We established
the current protocol with no a priori assumptions regard-
ing the protein composition of the calcifying EVs; how-
ever, we were able to detect many previously established
markers of calcifying matrix vesicles (Table III). Once we
have enriched our population of interest, we can continue
to perform advanced proteomic profiling of calcifying
vesicles using targeted mass spectrometry enabled by our
spectral library (Fig. 4b) that contains MS fingerprints for
all proteins enriched in this vesicle population (Fig. 4a).
These targeted proteomics approaches offer increased sen-
sitivity to better characterize subtle differences in protein
expression (33) and compare the calcifying matrix vesicles
produced by SMCs cultured in the conditions utilized
in this study to matrix vesicles from other calcifying cells
and those from calcified tissues.
In addition to speed and time, researchers should also
consider the effect of media density and viscosity and the
length of the ultracentrifugation tubes used to collect the
samples. Larger tubes or a more dense or viscous media
would require longer or higher speed ultracentrifuga-
tion protocols than shorter tubes or less viscous media (see
physical density analysis below). By considering all of
these parameters, studies may be able to better identify
protein changes in vesicle populations that can then
be used to establish more sensitive isolation techniques
(e.g. immunoprecipitation). Further, by understanding the
consequences and theoretical underpinnings of ultracen-
trifugation, important physical properties of vesicular
populations may be ascertained.
Density analysisThe ultracentrifugation methods presented can be used
to estimate differences in density between calcifying
and non-calcifying EVs. As shown, significantly greater
amounts of protein are pelleted after 10 min ultracen-
trifugation for the calcifying samples compared to the
control samples. If the amount of protein per vesicle is
approximately constant, the ratio of the protein pelleted
rates (Fig. 2f) should match the ratio of the initial pellet
rates measured by NTA (Fig. 2d). Taking the ratio of the
initial rates of vesicle depletion shown in Fig. 2d gives a
value of 1.37, which closely matches the ratio of protein
accumulation (Fig. 2e) of 1.35. In other words, the rate at
which vesicles are depleted in the calcifying samples over
the first 10 min (kcalcifying) is 1.35 times higher than the
rate at which vesicles are depleted from control samples
over the same time (kcontrol):
kcalcifying
kcontrol¼ 1:35 (1)
By considering the forces acting on vesicles during
ultracentrifugation, we can show that these results indi-
cate that calcifying vesicles are approximately 35% more
dense (i.e. reduced vesicle density, rv, compared to the
culture media density, rs) than control vesicles. At ter-
minal velocity as the vesicles are pelleted, the force
produced by ultracentrifugation (Fc) is opposed by a
buoyancy force (FB) and a drag force on the vesicle (FD).
FC ¼ FB þ FD (2)
The Fc is found by multiplying the mass of an indiv-
idual vesicle by the acceleration due to ultracentrifugation.
Fc ¼4
3pr3qvx
2R (3)
where r is the radius of an individual vesicle, rv is the
density of an individual vesicle, v is the angular velocity
of the centrifuge rotor and R is the radius from the edge
of the centrifuge arm to the centre of the rotor. The FB
is generated by the displacement of the liquid as the vesicle
moves through the solution with the ultracentrifugation
force.
FB ¼4
3pr3qsx
2R; (4)
where rs is the density of the culture media solution. At
terminal velocity (vt, when the acceleration of the vesicles
is zero), the FD experienced by a vesicle is a function of
the vesicle radius and the viscosity of the culture media
solution (h) such that
FD ¼ 6prgvt: (5)
As in equation (1), at terminal velocity, the FC is
balanced by the FD and FB to give
4
3pr3qvx
2R ¼ 4
3pr3qsx
2Rþ 6prgvt: (6)
By rearranging and combining terms, we find a
relationship for the density difference between the vesicle
and solution as a function of the other parameters.
qv � qsð Þ ¼ 9g
2r2x2Rvt: (7)
For the time required to pellet a vesicle (t) over a
certain length (l), since there is no acceleration when the
vesicle reaches vt,
vt ¼l
s: (8)
Table III. PSM of known proteins involved in calcification from
calcifying EVs
Donor 1 Donor 2
Protein 10 min 30 min 10 min 30 min
Annexin A2 44 23 53 36
Annexin A5 6 3 18 12
Annexin A6 � � 8 18
S100A9 5 � � �
Joshua D. Hutcheson et al.
10(page number not for citation purpose)
Citation: Journal of Extracellular Vesicles 2014, 3: 25129 - http://dx.doi.org/10.3402/jev.v3.25129
The time, t, required to pellet a vesicle is the reciprocal
of the rate at which vesicles are pelleted (k):
vt ¼ lk: (9)
Therefore, the original relationship for reduced density
becomes
qv � qsð Þ ¼ 9gl
2r2x2Rk: (10)
If we reasonably assume that all ultracentrifugation
parameters are constant, that the vesicles from each treat-
ment group are similar in size, and that the vesicles travel
similar lengths in the tubes, then the ratio of reduced
densities simplifies to
qv � qsð ÞOM
qv � qsð ÞNM
¼ kOM
kNM
: (11)
The calcifying and control subscripts denote the
reduced densities and rates for calcifying and control
samples, respectively. As shown in equation (1), from our
data this ratio of rates is approximately 1.35, and there-
fore, the ratio of the reduced densities must also equal
1.35. Using this approach, we can begin to estimate the
effects of hydroxyapatite mineralization on the physical
properties of the calcifying EVs. Future studies could
use spectroscopic techniques to further probe the mineral
content within the calcifying vesicles for better analyses
of the mass that is added to the vesicles by the growing
mineral. The nucleating events associated with calcifica-
tion remain largely unknown. By better understanding the
physical parameters, we can begin to build better models
of vesicle interactions and the nucleation of mineral asso-
ciated with calcification. Further, as shown in this study,
we can also take advantage of these physical changes
to separate the calcification for enhanced signal-to-noise
in our proteomic analyses and ultimately give a clearer
fingerprint of calcifying vesicles. These techniques may
lead to the identification of novel therapeutic approaches
to prevent or alter the nucleation of calcification by EVs.
Acknowledgements
The authors thank Mr. Iwao Yamada and Mr. Brett Pieper for mass
spectrometry support.
Conflict of interest and fundingThis project was supported by a research grant from Kowa
Company Ltd. to Dr. Masanori Aikawa. Dr. Masaya
Iwashita is an employee of Kowa Company Ltd. Dr. Elena
Aikawa is supported by grants from the National Institutes
of Health (R01HL114805; R01HL109506).
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